Energy sources in sperm capacitation media have various effects on mammalian sperm and are required for stimulation of hyperactivated motility and/or acrosome reactions in some species. The present experiments were performed to investigate the energy substrate requirement for macaque sperm capacitation. The capacitation status of sperm was assessed in terms of hyperactivated motility and the capability to acrosome react after binding to the macaque zona pellucida. Semen from 6 cynomolgus macaques was washed through 60% percoll, resuspended and washed with BWW media. In one set of experiments, sperm were incubated in the complete media or in media without glucose. In another set of experiments, the complete media was used for comparison with media containing no energy substrates. The absence of glucose did not affect survival of sperm during a 6 hr incubation period; however, removal of all energy substrates resulted in a decrease in percent motility by 3 hr. Sperm were incubated for 1 hr prior to evaluation of sperm motility by computer aided sperm analysis (CASA) and sperm-zona binding experiments. During the last 30 min of incubation, half of the aliquots of sperm suspensions were treated with activators (act, caffeine and DBCAMP, 1mm each). As previously reported, when sperm were incubated in complete media and treated with act there were changes in sperm movements that are consistent with hyperactivation. Similar or greater changes were observed in sperm that were incubated prior to treatment with act in glucose-free media or in media without any energy substrates. Whether sperm were incubated in complex media or in glucose-free media, sperm binding to zonae was enhanced when the sperm were treated with act. The absence of glucose in the incubation media had no deleterious effect on the zona-induced acrosome reaction, which could be demonstrated whether or not sperm were treated with act. Sperm incubated in media without energy substrates also were capable of binding to the zona pellucida and of undergoing the acrosome reaction following treatment with act. These results indicate that macaque sperm, like human sperm, do not require energy substrates in incubation media in order to undergo capacitation in vitro.